This invention relates to maintaining vertebrate liver cells in culture.
The vertebrate liver is a complex and indispensable organ that provides many vital functions, including metabolism, excretion, detoxification, storage, and phagocytosis. In humans, acute severe liver failure, such as acute fulminant hepatitis, results from massive hepatocellular necrosis caused by viruses, drugs, or chemicals, and can have a mortality exceeding 80%. Chronic liver failure in humans is most commonly the result of hepatocellular replacement by scar tissue or cirrhosis. Cirrhosis is the sixth leading cause of death in the United States and ranks eighth in economic cost among major illnesses; in patients over 40 years old, it is the fifth ranking cause of death.
There exists no satisfactory practical means for liver replacement other than transplantation.
Many approaches to replacing the detoxification function of the liver have been attempted, including nonbiological, biological, and semibiological or hybrid approaches, but few approaches except for whole organ transplantation have had even limited success.
Biological approaches to replacing the detoxification function of the liver have employed transplantation, cross circulation, exchange transfusion, and extracorporeal perfusion.
Human orthotopic liver transplantation for both acute fulminant hepatitis and chronic liver failure now has an actuarial survival of 80% with careful selection of donor and recipient pools. However, owing to donor scarcity and short preservation time of the donor liver, many patients continue to die without transplantation. Heterotopic auxiliary liver transplantation, i.e., emplacement of an additional liver in other than the normal location continues to be explored with limited success in improving survival.
Potential transmission of disease proscribes use of cross circulation between humans, and immunological consequences proscribes its use between a human and a nonhuman animal.
In extracorporeal perfusion, a heterologous liver is used to clear toxins in an extracorporeal circuit, but livers such as porcine or bovine livers degrade after 6 hours or less of such use, and baboon livers degrade within one day. More recent attempts to improve extracorporeal perfusion have included combining it with cross circulation, but with only limited success. These biological approaches have as disadvantages that the surgical techniques are complicated, the immunological consequences are complex, preservation of the livers is difficult, and a high risk exists for transmission of infectious agents such as hepatitis virus or human immunodeficiency virus.
Nonbiological approaches to replacing the detoxification function of the liver have included dialysis, hemoperfusion, and ion exchange.
Dialysis, which is effective in renal failure, has shown no beneficial effect in hepatic coma where membranes are used which remove molecules below 15,000 daltons. Hemoperfusion of blood through charcoal columns removes larger molecules than dialysis, particularly protein-bound toxins, and hemoperfusion may actually reduce mortality in acute fulminant hepatitis if therapy is initiated during Stages II or III, which is early in the onset of hepatic encephalopathy; such attempts have been ineffective after onset of irreversible cerebral edema in Stage IV. Passing blood through activated charcoal removes toxins causing hepatic coma, but in an initial clinical trial, overall survival rate was 24% compared to 18% without treatment. Problems with hemoperfusion involving charcoal-induced thrombogenicity and platelet activation have more recently been partly solved by coating charcoal with biocompatible materials, encapsulating the charcoal, perfusing with plasma instead of whole blood, and administering anti-platelet drugs such as prostacyclin. Both a temporary recovery of consciousness and improved survival have been reported with coated charcoal hemoperfusion in acetaminophen-induced fulminant liver failure in humans.
Although these studies have shown limited positive effects, non-biological methods are for the most part inadequate because of their monofocal approach. Major liver functions, such as, for example, metabolism, synthesis, and storage are ignored in these nonbiological systems. It is also likely that some toxins are left in the circulation while some salutory regeneration factors are removed.
Combinations of biological and nonbiological approaches into semibiological or hybrid approaches to replacing the detoxification function of the liver have utilized a combination of enzymes or cells or tissues with mechanical devices, such as immobilized enzymes, dialysis membranes with single cell hepatocyte suspensions or liver slices, and hepatocytes immobilized in alginate, or fetal hepatocyte cells growing on hollow fiber capillaries. Enzyme immobilization using enzymes important in liver function, and using charcoal, red cell ghosts, hollow fibers, and artificial cells as solid-phase supports, is limited in that only one substrate is altered with each such treatment, and it is too simplistic an approach for liver failure in view of the fact that hepatic coma appears to result from more than one different toxin.
An approach combining dialysis with liver pieces or single cell suspensions enclosed within a reactor through which blood is perfused have been effective in lowering toxin concentrations. In such systems, however, oxygen transfer and movement of protein-bound toxins is limited by diffusion and the friable consistency of the liver does not allow the preparation of slices sufficiently thin to overcome these diffusional limitations.
One approach to overcoming the diffusional limitations inherent in the systems described above involves transplanting a liver cell suspension into a site such as the peritoneal cavity, the spleen, and the lung. Syngeneic, allogeneic, and xenogeneic hepatocyte transplantations in animals have resulted in improved survival rates, but rejection of allogeneic or xenogeneic transplants is expected. In combination with immunosuppression, hepatocytes attached to microcarriers have been demonstrated to replace glucuronyl transferase activity in Gunn rats and albumin production in Nagase rats. Ideally, protection of the transplanted hepatocytes from graft rejection is desired. To this end, entrapment of hepatocytes in collagen, alginate, agarose, and urethane prepolymer has been tried using configurations such as spherical gel beads and cylindrical hollow fibers. However, these protective barriers can impose significant mass transfer resistances, and thus can limit the viability and/or function of the protected cells.
Hepatocytes are difficult to maintain in a viable condition, and hepatocytes maintained in culture lose their liver phenotype over short time periods. Hepatocytes are anchorage-dependent, highly differentiated cells that are difficult to maintain in vitro, Guguen-Guillouzo (1983), Molec. cell Biochem., Vol. 53/54, pp. 35-56; Reid, et al., (1984), Hepatology, 4(3), pp. 548-559. Early attempts to culture liver cells from organ explants invariably led to overgrowth of fibroblasts and undefined epithelial cell lines, Watanabe (1966), Exp. Cell Res., Vol. 42, pp. 685-699. Short-term cultures of hepatocytes became possible with the introduction of enzymatic dissociation of the liver, Berry et al. (1969), J. Cell Biol., Vol. 43, pp. 507-520, resulting in large numbers of cells that were mostly hepatocytes. Conventional culture configurations include cell suspensions in stirred flasks and cell monolayers on plastic dishes, Bissell et al. (1973), J. Cel Biol., Vol. 59, pp. 722-734, Phillips et al. (1974), Lab Inv., Vol. 31, pp. 533-542. Hepatocytes in suspension cultures cluster into large clumps of cells within one day of incubation, with rapid loss of function. Hepatocytes in monolayer cultures dedifferentiate and lose adult liver phenotype within a week of incubation. Generally speaking, these cultures tend to fetalize with age of culture, Leffert et (1978), Proc Natl. Acad. Sci., Vol. 75, pp. 1834-1838, expressing fetal pyruvate kinase isozymes or .alpha.-fetoprotein. These hepatocytes gradually die and eventually detach; concomitantly other cell types grow to overtake the culture.
More recently, efforts were made to culture hepatocytes in arginine-free media, Leffert et al. (1972), J. Cell Biol, Vol. 52, p. 559, on floating collagen membrane, Michalopoulos et al. (1975), Exp. Cell Res., Vol. 94, p. 70, on liver biomatrix, Reid et al. (1980), Ann. NY Acad. Sci., p. 70, along with other liver cells, Guguen-Guillouzo et al. (1983), Exp. Cell Res., Vol. 143, p. 47, and in the presence of dimethyl sulfoxide, Isom et al. (1984), PNAS, Vol. 82, p. 3252. In each of these approaches, liver-specific functions were shown to be maintained for periods ranging from 2 to 7 weeks. However, Clayton (1985), Molec. Cell Bio., Vol. 5, p. 2623, showed that none of these cultures exhibited normal liver specific transcriptional rate; the level of liver-specific mRNA was, at best, kept at a constant level by stabilizing the original mRNA.
Hepatoma cell lines and some liver-derived cell lines grow well in vitro, but it has been found that these cell lines often lack many liver-specific functions, Clayton et al., (1985), Molec. Cell Biol., Vol. 5, p. 2633, and the tumorigenic nature of these cells limits their application in clinical situations.
Several methodological approaches to improving both morphology and function of cultured hepatocytes have been reported including addition of extracellular matrix products, addition of other cell types, and use of different media formulations. Leffert (1972), J. Cell Biol., Vol. 52, pp. 559-568; Michalopoulos et al., (1975), Exp. Cell Res., Vol. 94, pp. 70-78; Reid L. M., et al. (1980), Ann. NY Acad. Sci., pp. 70-76; Guguen-Guillouzo et al. (1983), Exp. Cell Res., Vol. 143, pp. 47-54; Isom et al., (1985), Proc. Natl. Acad. Sci., Vol. 82, pp. 3252-3256. Except for the use of matrix components as a substrate for hepatocyte culture, these methods face serious limitations when clinical implementation is considered. For example, it would be difficult to maintain an arginine-free environment once the hepatocytes are used as an artificial liver support; DMSO toxicity limits its use in patients with liver failure; and introduction of undefined epithelial cell lines into patients is clinically unacceptable.
It has been shown that when a suspension of liver cells is seeded on a culture dish, cells tend to reorganize such that they reconstitute many histological landmarks such as the bile canaliculus, Wanson et al. (1977), J. Cell Biol., Vol. 77, pp. 858-877. However, under known culture conditions the cells maintain this "in vivo-like" configuration only for a short time, and thereafter lose their structural and metabolic character as liver cells.
It is known that extracellular matrix and cell--cell interaction can influence the behavior and differentiation of cells. Polarization of cultured cells in response to addition of extracellular matrix has been demonstrated in several instances. For example, after collagen is overlayed on a monolayer of mammary epithelial cells cultured on collagen gel, cells reorganize to form structures with their lumens directed away from the collagen, Hall, et al. (1982), Proc. Natl. Acad. Sci., Vol. 79, pp. 4672-4676. Such tube-like structures resemble the mammary ducts that are present in vivo. This, of course, is the natural configuration of mammary epithelial cells, which line ducts in a monolayer fashion. The formation of a flat monolayer on a dish is an artifact of the physical constraints imposed by the culture environment.
Addition of extracellular matrix products, such as collagen, to cultures of hepatocytes can somewhat improve the maintenance of differentiated functions.